Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. Until recently, the prevailing models suggested that nuclear extrusion occurs via asymmetric cytokinesis or by a modified apoptotic process. Our recent findings reveal, however, that enucleation is driven in large part by the formation, movement and subsequent coalescence of vacuoles at the junction of the nucleus and the cytoplasm. Here we propose to harness our new insights to improve enucleation of erythroblasts expanded in vitro and to further define the mechanisms that govern terminal differentiation. Our specific aims are to: 1) Determine how vesicle trafficking drives enucleation, 2) Identify novel cellular factors that regulate enucleation, and 3) optimize small molecules that induce enucleation. Our work will enhance our understanding of the final maturation of red cell development and optimization of ex vivo erythrocyte production.